br Materials and methods br Results

Materials and methods
Results
Discussion This was the first large study to quantify both total HIV DNA and integrated HIV DNA, which is the main persistent form of HIV [25,26], in samples collected during PHI, chronic infection, and at various time points until the AIDS stage, which was made possible by two large French cohorts. The ANRS-SEROCO cohort is one of the rare large historical cohorts that included patients in the 1990s who, at that time, remained largely untreated during their follow-up and who had available longitudinal frozen cell samples. One of the objectives of this study was to explore the link between the amount of stable proviruses and the risk of HIV disease progression, with the hypothesis that unstable unintegrated forms may have a lesser impact on long-term evolution. From the time of recent seroconversion, the amount of total HIV DNA was higher in rapidly progressing patients than in slower progressors, which agreed with its known predictive value for HIV disease progression [10,27]. Here, we report for the first time the predictive value of the amount of integrated HIV DNA, which appeared to be even more strongly predictive of the risk of developing clinical AIDS. Integrated HIV DNA is probably mostly responsible for the predictive value of total HIV DNA observed in previous studies, since this stable form of HIV persistence drives the course of infection [2,5]. Integrated HIV DNA is indeed the major source of the viral replication and the HIV RNA level reflects the proportion of cells containing this HIV stable form. Yet, the pathogenic impact of integrated HIV DNA is not only through the viral replication since its predictive value is independent of the HIV RNA level. Another potential means to drive the pathogenesis could be the production of transcripts and Tanshinone IIA receptor from defective integrated genomes, which do not result in HIV RNA viremia, but contribute to the immune activation, as previously described [28]. The concomitant study of total HIV DNA and integrated HIV DNA showed that the percentage of integrated forms is highly informative. Indeed, this percentage was already high in rapid progressors at a time point within 12 months from seroconversion and remained high during disease progression, confirming that early events determine the profile of a “rapid progressor” or “slower progressor”. Total HIV DNA being strongly predictive of progression towards AIDS in PHI [29], the few PHI patients with a high proportion of integrated forms might be at a particularly high risk of becoming rapid progressors. These data are in line with the very low levels of integrated HIV DNA observed in the unique group of elite suppressors compared to patients under efficient cART with similar total HIV DNA levels [30]. The low levels of integrated HIV DNA may be the consequence of more efficient specific cytotoxic T-lymphocyte (CTL) responses. CTLs could have preferentially destroyed cells harbouring integrated viral DNA, in which the viral transcription and translation into proteins is more efficient, eventually leading to the maintenance of lower proportions of integrated forms [31]. Rapid progressors may have less efficient CTL responses and therefore maintain higher levels of integrated HIV DNA. Studies examining total HIV DNA have shown that the establishment of reservoirs is a very early event in PHI [32]. Although integrated HIV DNA is included in total HIV DNA, concomitant study of these two markers showed that they exhibit very different kinetics in the first year of infection, where the first three months after infection stand apart from the following months and years. Integrated HIV DNA levels were found to be low for most patients during PHI, while total HIV DNA levels were particularly high at that time, which was consistent with previous data showing a peak during this stage [29,32]. We observed a slight short decrease in integrated HIV DNA levels after the first weeks of infection (Fiebig III-IV), which is in agreement with the results previously reported for 19 untreated PHI patients from whom samples were collected during earlier stages (17 at Fiebig I-II stages), which showed a peak of integrated HIV DNA in week 2 and a decrease until week 6 after enrolment [18]. Ananworanich's work presented some results describing the evolution of total HIV DNA and integrated HIV DNA for ten patients during 144 weeks [18]. However, only the median results were presented for these two biomarkers and the proportions of stable forms among total HIV DNA and their evolution over time were not detailed for the different individuals. Thanks to a far greater number of patients and longer follow-up time, we showed here a more extensive increase of the proportion of integrated forms than in this previous work. With techniques allowing the comparison between the levels of total HIV DNA and integrated HIV DNA, we show the benefit of studying the percentage of integrated forms, their heterogeneity between patients and their impact on the outcome of untreated HIV infection. Although several PHI patients had undetectable integrated HIV DNA levels, the technique used here has a low quantification threshold and we are confident these results reflect truly low levels of integrated forms. The longer follow-up time also allowed us to highlight the progressive increase of total HIV DNA, which was not significant in previous shorter studies.